Wild rocket varieties &#39;prudenzia&#39; and &#39;anastazia&#39;

ABSTRACT

New wild rocket varieties designated ‘Prudenzia’ and ‘Anastazia’ are described. ‘Prudenzia’ and ‘Anastazia’ are wild rocket varieties exhibiting stability and uniformity.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/276,372, filed Jan. 8, 2016, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding. Inparticular, this invention relates to new wild rocket, Diplotaxistenuifolia varieties designated ‘Prudenzia’ and ‘Anastazia’.

BACKGROUND OF THE INVENTION

Wild rocket, Diplotaxis tenuifolia, is a specialty leaf vegetable in theBrassicaceae family. Wild rocket is also known as arugula, sand mustard,sand rocket, Lincoln's weed, white rocket, and wall rocket. Wild rocketis a flowering plant that is a fast growing, cool season crop andflowers under long days and high temperatures. Unlike lettuce, arugulais not bitter and the branches are often used as an ingredient insalads.

Wild rocket is native to Europe and Western Asia. However, wild rocketmay be found in temperate zones throughout much of the world. Wildrocket is an erect mustard-like plant with branching stems that canexceed half a meter in height. Yellow flowers grow from the top ofbranches of the stems, and the fruit is a straight, flat silique thatcan be up to five centimeters long. Wild rocket also has long leavesthat can be lobbed, and the foliage is aromatic when crushed. Wildrocket generally grows in clumps on the ground in various habitats.

Wild rocket is an important and valuable vegetable crop. Accordingly,there is a need for new wild rocket varieties. In particular, there is aneed for improved flat wild rocket varieties that are stable, highyielding, and agronomically sound.

SUMMARY OF THE INVENTION

In order to meet these needs, the present invention is directed toimproved wild rocket varieties.

As used herein wild rocket variety ‘Prudenzia’ is the same wild rocketvariety as wild rocket variety ‘E13R3007’ having ATCC Accession NumberX1 and disclosed in U.S. Provisional Application No. 62/276,372. Whilethe name has changed, wild rocket variety ‘Prudenzia’ has all thedefining characteristics of wild rocket variety ‘E13R3007’.

In one embodiment, the present invention is directed to wild rocket,Diplotaxis tenuifolia, seed designated as ‘Prudenzia’, representativesample of seed having been deposited under NCIMB Accession Number X1. Inone embodiment, the present invention is directed to a Diplotaxistenuifolia wild rocket plant and parts isolated therefrom produced bygrowing ‘Prudenzia’ wild rocket seed. In another embodiment, the presentinvention is directed to a Diplotaxis tenuifolia plant and partsisolated therefrom having all the physiological and morphologicalcharacteristics of a Diplotaxis tenuifolia plant produced by growing‘Prudenzia’ wild rocket seed having NCIMB Accession Number X1. In stillanother embodiment, the present invention is directed to an F₁ hybridDiplotaxis tenuifolia wild rocket seed, plants grown from the seed, anda head isolated therefrom having ‘Prudenzia’ as a parent, where‘Prudenzia’ is grown from ‘Prudenzia’ wild rocket seed having NCIMBAccession Number X1.

Wild rocket plant parts include wild rocket stems, wild rocket leaves,parts of wild rocket leaves, pollen, ovules, flowers, roots, cells, andthe like. In another embodiment, the present invention is furtherdirected to wild rocket stems, wild rocket leaves, parts of wild rocketleaves, flowers, pollen, and ovules, roots, or cells isolated from‘Prudenzia’ wild rocket plants. In another embodiment, the presentinvention is further directed to tissue culture of ‘Prudenzia’ wildrocket plants, and to wild rocket plants regenerated from the tissueculture, where the plant has all of the morphological and physiologicalcharacteristics of ‘Prudenzia’ wild rocket plants.

In still another embodiment, the present invention is further directedto packaging material containing ‘Prudenzia’ plant parts. Such packagingmaterial includes but is not limited to boxes, plastic bags, etc. The‘Prudenzia’ plant parts may be combined with other plant parts of otherplant varieties.

In yet another embodiment, the present invention is further directed toa method of selecting wild rocket plants, by a) growing ‘Prudenzia’ wildrocket plants where the ‘Prudenzia’ plants are grown from wild rocketseed having NCIMB Accession Number X1 and b) selecting a plant from stepa). In another embodiment, the present invention is further directed towild rocket plants, plant parts and seeds produced by the wild rocketplants where the wild rocket plants are isolated by the selection methodof the invention. In another embodiment, the present invention isfurther directed to a method of breeding wild rocket plants by crossinga wild rocket plant with a plant grown from ‘Prudenzia’ wild rocket seedhaving NCIMB Accession Number X1. In still another embodiment, thepresent invention is further directed to wild rocket plants, wild rocketparts from the wild rocket plants, and seeds produced therefrom wherethe wild rocket plant is isolated by the breeding method of theinvention. In some embodiments, the wild rocket plant isolated by thebreeding method is a transgenic wild rocket plant. In anotherembodiment, the present invention is directed to methods for producing awild rocket plant containing in its genetic material one or moretransgenes and to the transgenic wild rocket plant produced by thosemethods. In another embodiment, the present invention is directed tomethods for producing a male sterile wild rocket plant by introducing anucleic acid molecule that confers male sterility into a wild rocketplant produced by growing ‘Prudenzia’ wild rocket seed, and to malesterile wild rocket plants produced by such methods.

In another embodiment, the present invention is directed to methods ofproducing an herbicide resistant wild rocket plant by introducing a geneconferring herbicide resistance into a wild rocket plant produced bygrowing ‘Prudenzia’ wild rocket seed, where the gene is selected fromglyphosate, sulfonylurea, imidazolinone, dicamba, glufosinate, phenoxyproprionic acid, L-phosphinothricin, cyclohexone, cyclohexanedione,triazine, and benzonitrile. Certain embodiments are also directed toherbicide resistant wild rocket plants produced by such methods. Inanother embodiment, the present invention is directed to methods ofproducing a pest or insect resistant wild rocket plan by introducing agene conferring pest or insect resistance into a wild rocket plantproduced by growing ‘Prudenzia’ wild rocket seed, and to pest or insectresistant wild rocket plants produced by such methods. In certainembodiments, the gene conferring pest or insect resistance encodes aBacillus thuringiensis endotoxin. In another embodiment, the presentinvention is directed to methods of producing a disease resistant wildrocket plant by introducing a gene conferring disease resistance into awild rocket plant produced by growing ‘Prudenzia’ wild rocket seed, andto disease resistant wild rocket plants produced by such methods. Inanother embodiment, the present invention is directed to methods ofproducing a wild rocket plant with a value-added trait by introducing agene conferring a value-added trait into a wild rocket plant produced bygrowing ‘Prudenzia’ wild rocket seed, where the gene encodes a proteinselected from a ferritin, a nitrate reductase, and a monellin. Certainembodiments are also directed to wild rocket plants having a value-addedtrait produced by such methods.

In another embodiment, the present invention is directed to methods ofintroducing a desired trait into wild rocket variety ‘Prudenzia’, by:(a) crossing a ‘Prudenzia’ plant, where a sample of ‘Prudenzia’ wildrocket seed was deposited under NCIMB Accession Number X1, with a plantof another wild rocket variety that contains a desired trait to produceprogeny plants, where the desired trait is selected from male sterility;herbicide resistance; insect or pest resistance; modified bolting; andresistance to bacterial disease, fungal disease or viral disease; (b)selecting one or more progeny plants that have the desired trait; (c)backcrossing the selected progeny plants with a ‘Prudenzia’ plant toproduce backcross progeny plants; (d) selecting for backcross progenyplants that have the desired trait and all of the physiological andmorphological characteristics of wild rocket variety ‘Prudenzia’; and(e) repeating steps (c) and (d) two or more times in succession toproduce selected third or higher backcross progeny plants that comprisethe desired trait. Certain embodiments are also directed to wild rocketplants produced by such methods, where the plants have the desired traitand all of the physiological and morphological characteristics of wildrocket variety ‘Prudenzia’. In certain embodiments, the desired trait isherbicide resistance and the resistance is conferred to an herbicideselected from glyphosate, sulfonylurea, imidazolinone, dicamba,glufosinate, phenoxy proprionic acid, L-phosphinothricin, cyclohexone,cyclohexanedione, triazine, and benzonitrile. In other embodiments, thedesired trait is insect or pest resistance and the insect or pestresistance is conferred by a transgene encoding a Bacillus thuringiensisendotoxin.

In another embodiment, the present invention provides for single geneconverted plants of ‘Prudenzia’. The single transferred gene maypreferably be a dominant or recessive allele. Preferably, the singletransferred gene will confer such traits as male sterility, herbicideresistance, insect or pest resistance, modified fatty acid metabolism,modified carbohydrate metabolism, resistance for bacterial, fungal, orviral disease, male fertility, enhanced nutritional quality, andindustrial usage. The single gene may be a naturally occurring wildrocket gene or a transgene introduced through genetic engineeringtechniques.

In a further embodiment, the present invention relates to methods fordeveloping wild rocket plants in a wild rocket plant breeding programusing plant breeding techniques including recurrent selection,backcrossing, pedigree breeding, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selection, andtransformation. Seeds, wild rocket plants, and parts thereof, producedby such breeding methods are also part of the invention.

As used herein wild rocket variety ‘Anastazia’ is the same wild rocketvariety as wild rocket variety ‘E13R8011’ having ATCC Accession NumberX2 and disclosed in U.S. Provisional Application No. 62/276,372. Whilethe name has changed, wild rocket variety ‘Anastazia’ has all thedefining characteristics of wild rocket variety ‘E13R8011’.

In another embodiment, the present invention is directed to wild rocket,Diplotaxis tenuifolia, seed designated as ‘Anastazia’, representativesample of seed having been deposited under NCIMB Accession Number X2. Inone embodiment, the present invention is directed to a Diplotaxistenuifolia wild rocket plant and parts isolated therefrom produced bygrowing ‘Anastazia’ wild rocket seed. In another embodiment, the presentinvention is directed to a Diplotaxis tenuifolia plant and partsisolated therefrom having all the physiological and morphologicalcharacteristics of a Diplotaxis tenuifolia plant produced by growing‘Anastazia’ wild rocket seed having NCIMB Accession Number X2. In stillanother embodiment, the present invention is directed to an F₁ hybridDiplotaxis tenuifolia wild rocket seed, plants grown from the seed, anda head isolated therefrom having ‘Anastazia’ as a parent, where‘Anastazia’ is grown from ‘Anastazia’ wild rocket seed having NCIMBAccession Number X2.

Wild rocket plant parts include wild rocket stems, wild rocket leaves,parts of wild rocket leaves, pollen, ovules, flowers, roots, cells, andthe like. In another embodiment, the present invention is furtherdirected to wild rocket stems, wild rocket leaves, parts of wild rocketleaves, flowers, pollen, and ovules, roots, or cells isolated from‘Anastazia’ wild rocket plants. In another embodiment, the presentinvention is further directed to tissue culture of ‘Anastazia’ wildrocket plants, and to wild rocket plants regenerated from the tissueculture, where the plant has all of the morphological and physiologicalcharacteristics of ‘Anastazia’ wild rocket plants.

In still another embodiment, the present invention is further directedto packaging material containing ‘Anastazia’ plant parts. Such packagingmaterial includes but is not limited to boxes, plastic bags, etc. The‘Anastazia’ plant parts may be combined with other plant parts of otherplant varieties.

In yet another embodiment, the present invention is further directed toa method of selecting wild rocket plants, by a) growing ‘Anastazia’ wildrocket plants where the ‘Anastazia’ plants are grown from wild rocketseed having NCIMB Accession Number X2 and b) selecting a plant from stepa). In another embodiment, the present invention is further directed towild rocket plants, plant parts and seeds produced by the wild rocketplants where the wild rocket plants are isolated by the selection methodof the invention. In another embodiment, the present invention isfurther directed to a method of breeding wild rocket plants by crossinga wild rocket plant with a plant grown from ‘Anastazia’ wild rocket seedhaving NCIMB Accession Number X2. In still another embodiment, thepresent invention is further directed to wild rocket plants, wild rocketparts from the wild rocket plants, and seeds produced therefrom wherethe wild rocket plant is isolated by the breeding method of theinvention. In some embodiments, the wild rocket plant isolated by thebreeding method is a transgenic wild rocket plant. In anotherembodiment, the present invention is directed to methods for producing awild rocket plant containing in its genetic material one or moretransgenes and to the transgenic wild rocket plant produced by thosemethods. In another embodiment, the present invention is directed tomethods for producing a male sterile wild rocket plant by introducing anucleic acid molecule that confers male sterility into a wild rocketplant produced by growing ‘Anastazia’ wild rocket seed, and to malesterile wild rocket plants produced by such methods.

In another embodiment, the present invention is directed to methods ofproducing an herbicide resistant wild rocket plant by introducing a geneconferring herbicide resistance into a wild rocket plant produced bygrowing ‘Anastazia’ wild rocket seed, where the gene is selected fromglyphosate, sulfonylurea, imidazolinone, dicamba, glufosinate, phenoxyproprionic acid, L-phosphinothricin, cyclohexone, cyclohexanedione,triazine, and benzonitrile. Certain embodiments are also directed toherbicide resistant wild rocket plants produced by such methods. Inanother embodiment, the present invention is directed to methods ofproducing a pest or insect resistant wild rocket plan by introducing agene conferring pest or insect resistance into a wild rocket plantproduced by growing ‘Anastazia’ wild rocket seed, and to pest or insectresistant wild rocket plants produced by such methods. In certainembodiments, the gene conferring pest or insect resistance encodes aBacillus thuringiensis endotoxin. In another embodiment, the presentinvention is directed to methods of producing a disease resistant wildrocket plant by introducing a gene conferring disease resistance into awild rocket plant produced by growing ‘Anastazia’ wild rocket seed, andto disease resistant wild rocket plants produced by such methods. Inanother embodiment, the present invention is directed to methods ofproducing a wild rocket plant with a value-added trait by introducing agene conferring a value-added trait into a wild rocket plant produced bygrowing ‘Anastazia’ wild rocket seed, where the gene encodes a proteinselected from a ferritin, a nitrate reductase, and a monellin. Certainembodiments are also directed to wild rocket plants having a value-addedtrait produced by such methods.

In another embodiment, the present invention is directed to methods ofintroducing a desired trait into wild rocket variety ‘Anastazia’, by:(a) crossing a ‘Anastazia’ plant, where a sample of ‘Anastazia’ wildrocket seed was deposited under NCIMB Accession Number X2, with a plantof another wild rocket variety that contains a desired trait to produceprogeny plants, where the desired trait is selected from male sterility;herbicide resistance; insect or pest resistance; modified bolting; andresistance to bacterial disease, fungal disease or viral disease; (b)selecting one or more progeny plants that have the desired trait; (c)backcrossing the selected progeny plants with a ‘Anastazia’ plant toproduce backcross progeny plants; (d) selecting for backcross progenyplants that have the desired trait and all of the physiological andmorphological characteristics of wild rocket variety ‘Anastazia’; and(e) repeating steps (c) and (d) two or more times in succession toproduce selected third or higher backcross progeny plants that comprisethe desired trait. Certain embodiments are also directed to wild rocketplants produced by such methods, where the plants have the desired traitand all of the physiological and morphological characteristics of wildrocket variety ‘Anastazia’. In certain embodiments, the desired trait isherbicide resistance and the resistance is conferred to an herbicideselected from glyphosate, sulfonylurea, imidazolinone, dicamba,glufosinate, phenoxy proprionic acid, L-phosphinothricin, cyclohexone,cyclohexanedione, triazine, and benzonitrile. In other embodiments, thedesired trait is insect or pest resistance and the insect or pestresistance is conferred by a transgene encoding a Bacillus thuringiensisendotoxin.

In another embodiment, the present invention provides for single geneconverted plants of ‘Anastazia’. The single transferred gene maypreferably be a dominant or recessive allele. Preferably, the singletransferred gene will confer such traits as male sterility, herbicideresistance, insect or pest resistance, modified fatty acid metabolism,modified carbohydrate metabolism, resistance for bacterial, fungal, orviral disease, male fertility, enhanced nutritional quality, andindustrial usage. The single gene may be a naturally occurring wildrocket gene or a transgene introduced through genetic engineeringtechniques.

In a further embodiment, the present invention relates to methods fordeveloping wild rocket plants in a wild rocket plant breeding programusing plant breeding techniques including recurrent selection,backcrossing, pedigree breeding, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selection, andtransformation. Seeds, wild rocket plants, and parts thereof, producedby such breeding methods are also part of the invention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference bystudy of the following description.

DETAILED DESCRIPTION OF THE INVENTION

There are numerous steps in the development of novel, desirable wildrocket germplasm. Plant breeding begins with the analysis of problemsand weaknesses of current wild rocket germplasms, the establishment ofprogram goals, and the definition of specific breeding objectives. Thenext step is selection of germplasm that possess the traits to meet theprogram goals. The goal is to combine in a single variety or hybrid animproved combination of desirable traits from the parental germplasm.These important traits may include increased head size and weight,higher seed yield, improved color, resistance to diseases and insects,tolerance to drought and heat, and better agronomic quality.

Choice of breeding or selection methods can depend on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of variety used commercially (e.g., F₁ hybrid variety, purelinevariety, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable variety. This approach hasbeen used extensively for breeding disease-resistant varieties. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program may include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, and can include gain fromselection per year based on comparisons to an appropriate standard, theoverall value of the advanced breeding lines, and the number ofsuccessful varieties produced per unit of input (e.g., per year, perdollar expended, etc.).

Promising advanced breeding lines may be thoroughly tested and comparedto appropriate standards in environments representative of thecommercial target area(s) for at least three years. The best lines canthen be candidates for new commercial varieties. Those still deficientin a few traits may be used as parents to produce new populations forfurther selection. These processes, which lead to the final step ofmarketing and distribution, may take from ten to twenty years from thetime the first cross or selection is made.

One goal of wild rocket plant breeding is to develop new, unique, andgenetically superior wild rocket varieties. A breeder can initiallyselect and crosses two or more parental lines, followed by repeatedselfing and selection, producing many new genetic combinations.Moreover, a breeder can generate multiple different genetic combinationsby crossing, selfing, and mutations. A plant breeder can then selectwhich germplasms to advance to the next generation. These germplasms maythen be grown under different geographical, climatic, and soilconditions, and further selections can be made during, and at the endof, the growing season.

The development of commercial wild rocket varieties thus requires thedevelopment of parental wild rocket varieties, the crossing of thesevarieties, and the evaluation of the crosses. Pedigree breeding andrecurrent selection breeding methods may be used to develop varietiesfrom breeding populations. Breeding programs can be used to combinedesirable traits from two or more varieties or various broad-basedsources into breeding pools from which new varieties are developed byselfing and selection of desired phenotypes. The new varieties arecrossed with other varieties and the hybrids from these crosses areevaluated to determine which have commercial potential.

Pedigree breeding is generally used for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population. Then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new varieties.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding may be used to transfer genes for a simply inherited,highly heritable trait into a desirable homozygous cultivar or line thatis the recurrent parent. The source of the trait to be transferred iscalled the donor parent. The resulting plant is expected to have theattributes of the recurrent parent (e.g., cultivar) and the desirabletrait transferred from the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineswith each generation due to failure of some seeds to germinate or someplants to produce at least one seed. As a result, not all of the F₂plants originally sampled in the population will be represented by aprogeny when generation advance is completed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques known in the artthat are available for the analysis, comparison and characterization ofplant genotype. Such techniques include, without limitation, IsozymeElectrophoresis, Restriction Fragment Length Polymorphisms (RFLPs),Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs), AmplifiedFragment Length polymorphisms (AFLPs), Simple Sequence Repeats (SSRs,which are also referred to as Microsatellites), and Single NucleotidePolymorphisms (SNPs).

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Mutation breeding may also be used to introduce new traits into wildrocket varieties. Mutations that occur spontaneously or are artificiallyinduced can be useful sources of variability for a plant breeder. Thegoal of artificial mutagenesis is to increase the rate of mutation for adesired characteristic. Mutation rates can be increased by manydifferent means including temperature, long-term seed storage, tissueculture conditions, radiation (such as X-rays, Gamma rays, neutrons,Beta radiation, or ultraviolet radiation), chemical mutagens (such asbase analogs like 5-bromo-uracil), antibiotics, alkylating agents (suchas sulfur mustards, nitrogen mustards, epoxides, ethyleneamines,sulfates, sulfonates, sulfones, or lactones), azide, hydroxylamine,nitrous acid, or acridines. Once a desired trait is observed throughmutagenesis the trait may then be incorporated into existing germplasmby traditional breeding techniques. Details of mutation breeding can befound in Principles of Cultivar Development by Fehr, MacmillanPublishing Company (1993).

The production of double haploids can also be used for the developmentof homozygous varieties in a breeding program. Double haploids areproduced by the doubling of a set of chromosomes from a heterozygousplant to produce a completely homozygous individual. For example, seeWan, et al., Theor. Appl. Genet., 77:889-892 (1989).

Additional non-limiting examples of breeding methods that may be usedinclude, without limitation, those found in Principles of PlantBreeding, John Wiley and Son, pp. 115-161 (1960); Allard (1960);Simmonds (1979); Sneep, et al. (1979); Fehr (1987); and “Carrots andRelated Vegetable Umbelliferae,” Rubatzky, V. E., et al. (1999).

Definitions

In the description that follows, a number of terms are used. In order toprovide a clear and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided:

Allele. The allele is any of one or more alternative forms of a gene,all of which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Bolting. The premature development of a flowering stalk, and subsequentseed, before a plant produces a food crop. Bolting is typically causedby late planting when temperatures are low enough to cause vernalizationof the plants.

Bremia lactucae. An oomycete that causes downy mildew in leafyvegetables, such as lettuce and wild rocket, in cooler growing regions.

Cotyledon. One of the first leaves of the embryo of a seed plant;typically one or more in monocotyledons, two in dicotyledons, and two ormore in gymnosperms.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

First water date. The date the seed first receives adequate moisture togerminate. This can and often does equal the planting date.

Gene. As used herein, “gene” refers to a segment of nucleic acid. A genecan be introduced into a genome of a species, whether from a differentspecies or from the same species, using transformation or variousbreeding methods.

Maturity date. Maturity refers to the stage when the plants are of fullsize or optimum weight, in marketable form or shape to be of commercialor economic value.

Quantitative Trait Loci. Quantitative Trait Loci (QTL) refers to geneticloci that control to some degree, numerically representable traits thatare usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Horticulture Society Enterprise Ltd., RHS Garden;Wisley, Woking; Surrey GU236QB, UK.

Single gene converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing or via genetic engineering where essentially all of thedesired morphological and physiological characteristics of a line arerecovered in addition to the single gene transferred into the line viathe backcrossing technique or via genetic engineering.

Overview of the Variety ‘Prudenzia’

Wild rocket variety ‘Prudenzia’ is a hybrid wild rocket suitable forgreenhouse, open field, and tunnel production. Wild rocket variety‘Prudenzia’ was chosen for its long leaf length, broad leaf width,strong leaf division, dark green leaf color, late time of flowering, andintermediate resistance to downy mildew (Peronospora parasitica).

The variety has shown uniformity and stability for the traits, withinthe limits of environmental influence for the traits. It has beenincreased with continued observation for uniformity. No variant traitshave been observed or are expected in variety ‘Prudenzia’.

Objective Description of the Variety ‘Prudenzia’

Wild rocket variety ‘Prudenzia’ has the following morphologic and othercharacteristics:

Leaves:

Attitude: Semi erect; similar to variety ‘Discovery’

Color of blade: Green; similar to variety ‘Nature’

Intensity of color: Dark

Length: Long; similar to variety ‘Nature’

Width: Broad; similar to variety ‘Nature’

Division: Strong

Width of primary lobes: Medium

Secondary lobing: Medium; similar to variety ‘Nature’

Plant:

Time of flowering: Late; similar to variety ‘Nature’

Height at flowering stage: Long; similar to varieties ‘Verdia’ and‘Tiger’

Disease/Pest Resistance:

Downy Mildew (Peronospora parasitica): Intermediate resistance

Comparison of ‘Prudenzia’ to Commercial Wild Rocket Variety

Table 1 below compares some of the characteristics of wild rocketvariety ‘Prudenzia’ with the wild rocket variety ‘Nature’. Column 1lists the characteristics, column 2 shows the characteristics for wildrocket variety ‘Prudenzia’, and column 3 shows the characteristics forwild rocket variety ‘Nature’.

TABLE 1 Characteristic ‘Prudenzia’ ‘Nature’ Leaf intensity of color DarkLight

Table 2 below compares some of the characteristics of wild rocketvariety ‘Prudenzia’ with the wild rocket variety ‘Bellezia’. Column 1lists the characteristics, column 2 shows the characteristics for wildrocket variety ‘Prudenzia’, and column 3 shows the characteristics forwild rocket variety ‘Bellezia’.

TABLE 2 Characteristic ‘Prudenzia’ ‘Bellezia’ Leaf length Long Short

Overview of the Variety ‘Anastazia’

Wild rocket variety ‘Anastazia’ is a wild rocket variety suitable forgreenhouse, open field, and tunnel production. It is the result ofnumerous generations of plant selections chosen for its yield, shortleaf length, medium leaf width, weak leaf division, late flowering, andintermediate resistance to downy mildew (Peronospora parasitica).

The variety has shown uniformity and stability for the traits, withinthe limits of environmental influence for the traits. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The line has been increased withcontinued observation for uniformity. No variant traits have beenobserved or are expected in variety ‘Anastazia’.

Objective Description of the Variety ‘Anastazia’

Wild rocket variety ‘Anastazia’ has the following morphologic and othercharacteristics:

Leaves:

Attitude: Erect; similar to variety ‘Olivetta’

Color of blade: Green; similar to variety ‘Nature’

Intensity of color: Dark

Length: Short; similar to variety ‘Olivetta’

Width: Medium; similar to variety ‘Tiger’

Division: Weak; similar to variety ‘Tiger’

Width of primary lobes: Medium

Secondary lobing: Absent or weak; similar to variety ‘Tiger’

Plant:

Time of flowering: Late; similar to variety ‘Nature’

Height at flowering stage: Short; similar to variety ‘Tiger’

Disease/Pest Resistance:

Downy Mildew (Peronospora parasitica): Intermediate resistance

Comparison of ‘Anastazia’ to Commercial Wild Rocket Variety

Table 3 below compares some of the characteristics of wild rocketvariety ‘Anastazia’ with the wild rocket variety ‘Bellezia’. Column 1lists the characteristics, column 2 shows the characteristics for wildrocket variety ‘Anastazia’, and column 3 shows the characteristics forwild rocket variety ‘Bellezia’.

TABLE 3 Characteristic ‘Anastazia’ ‘Bellezia’ Division of leaf WeakMedium

Table 4 below compares some of the characteristics of wild rocketvariety ‘Anastazia’ with the wild rocket variety ‘Tiger’. Column 1 liststhe characteristics, column 2 shows the characteristics for wild rocketvariety ‘Anastazia’, and column 3 shows the characteristics for wildrocket variety ‘Tiger’.

TABLE 4 Characteristic ‘Anastazia’ ‘Tiger’ Time of flowering Late Medium

Further Embodiments

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method, suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably, expression vectors are introduced intoplant tissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformed plants obtained with the protoplasm of the invention areintended to be within the scope of this invention.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Any DNA sequences,whether from a different species or from the same species, which areintroduced into the genome using transformation or various breedingmethods, are referred to herein collectively as “transgenes.” Over thelast fifteen to twenty years, several methods for producing transgenicplants have been developed, and the present invention, in particularembodiments, also relates to transformed versions of the claimed line.

Nucleic acids or polynucleotides refer to RNA or DNA that is linear orbranched, single or double stranded, or a hybrid thereof. The term alsoencompasses RNA/DNA hybrids. These terms also encompass untranslatedsequence located at both the 3′ and 5′ ends of the coding region of thegene: at least about 1000 nucleotides of sequence upstream from the 5′end of the coding region and at least about 200 nucleotides of sequencedownstream from the 3′ end of the coding region of the gene. Less commonbases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine,and others can also be used for antisense, dsRNA, and ribozyme pairing.For example, polynucleotides that contain C-5 propyne analogues ofuridine and cytidine have been shown to bind RNA with high affinity andto be potent antisense inhibitors of gene expression. Othermodifications, such as modification to the phosphodiester backbone, orthe 2′-hydroxy in the ribose sugar group of the RNA can also be made.The antisense polynucleotides and ribozymes can consist entirely ofribonucleotides, or can contain mixed ribonucleotides anddeoxyribonucleotides. The polynucleotides of the invention may beproduced by any means, including genomic preparations, cDNApreparations, in vitro synthesis, RT-PCR, and in vitro or in vivotranscription.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector contains DNA thatcontains a gene under control of, or operatively linked to, a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed wild rocketplants using transformation methods as described below to incorporatetransgenes into the genetic material of the wild rocket plant(s).

Expression Vectors for Wild Rocket Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (for example, a promoter) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley, et al., PNAS, 80:4803 (1983).Another commonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen, et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford, et al., Plant Physiol.,86:1216 (1988); Jones, et al., Mol. Gen. Genet., 210:86 (1987); Svab, etal., Plant Mol. Biol., 14:197 (1990); Hille, et al., Plant Mol. Biol.,7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate, or bromoxynil. Comai, etal., Nature, 317:741-744 (1985); Gordon-Kamm, et al., Plant Cell,2:603-618 (1990); and Stalker, et al., Science, 242:419-423 (1988).

Selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase, and plantacetolactate synthase. Eichholtz, et al., Somatic Cell Mol. Genet.,13:67 (1987); Shah, et al., Science, 233:478 (1986); and Charest, etal., Plant Cell Rep., 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include α-glucuronidase (GUS),α-galactosidase, luciferase, chloramphenicol, and acetyltransferase.Jefferson, R. A., Plant Mol. Biol., 5:387 (1987); Teeri, et al., EMBOJ., 8:343 (1989); Koncz, et al., PNAS, 84:131 (1987); and DeBlock, etal., EMBO J., 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissues are available. Molecular Probes,Publication 2908, IMAGENE GREEN, pp. 1-4 (1993) and Naleway, et al., J.Cell Biol., 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of GUS genes asselectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie, et al., Science, 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Wild Rocket Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence containing a regulatory element (for example, a promoter).Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific.” A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters:

An inducible promoter is operably linked to a gene for expression inwild rocket. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in wild rocket. With an inducible promoter, therate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward,et al., Plant Mol. Biol., 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Meft, et al., PNAS, 90:4567-4571 (1993)); In2gene from maize which responds to benzenesulfonamide herbicide safeners(Hershey, et al., Mol. Gen. Genet., 227:229-237 (1991) and Gatz, et al.,Mol. Gen. Genet., 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz, etal., Mol. Gen. Genet., 227:229-237 (1991)). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena,et al., PNAS, 88:0421 (1991).

B. Constitutive Promoters:

A constitutive promoter is operably linked to a gene for expression inwild rocket or the constitutive promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in wild rocket.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell, et al., Nature, 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy, et al., Plant Cell, 2:163-171 (1990));ubiquitin (Christensen, et al., Plant Mol. Biol., 12:619-632 (1989) andChristensen, et al., Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last,et al., Theor. Appl. Genet., 81:581-588 (1991)); MAS (Velten, et al.,EMBO J., 3:2723-2730 (1984)) and maize H3 histone (Lepetit, et al., Mol.Gen. Genet., 231:276-285 (1992) and Atanassova, et al., Plant J., 2(3):291-300 (1992)). The ALS promoter, Xbal/Ncol fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said Xbal/Ncol fragment), represents a particularly usefulconstitutive promoter. See PCT Application No. WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters:

A tissue-specific promoter is operably linked to a gene for expressionin wild rocket. Optionally, the tissue-specific promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in wild rocket. Plantstransformed with a gene of interest operably linked to a tissue-specificpromoter produce the protein product of the transgene exclusively, orpreferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Mural, et al., Science, 23:476-482(1983) and Sengupta-Gopalan, et al., PNAS, 82:3320-3324 (1985)); aleaf-specific and light-induced promoter such as that from cab orrubisco (Simpson, et al., EMBO J., 4(11):2723-2729 (1985) and Timko, etal., Nature, 318:579-582 (1985)); an anther-specific promoter such asthat from LAT52 (Twell, et al., Mol. Gen. Genet., 217:240-245 (1989)); apollen-specific promoter such as that from Zm13 (Guerrero, et al., Mol.Gen. Genet., 244:161-168 (1993)) or a microspore-preferred promoter suchas that from apg (Twell, et al., Sex. Plant Reprod., 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol., 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley,” Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., PlantPhysiol., 91:124-129 (1989); Fontes, et al., Plant Cell, 3:483-496(1991); Matsuoka, et al., PNAS, 88:834 (1991); Gould, et al., J. Cell.Biol., 108:1657 (1989); Creissen, et al., Plant J., 2:129 (1991);Kalderon, et al., A short amino acid sequence able to specify nuclearlocation, Cell, 39:499-509 (1984); and Steifel, et al., Expression of amaize cell wall hydroxyproline-rich glycoprotein gene in early leaf androot vascular differentiation, Plant Cell, 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is wild rocket. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR, and SSR analysis, which identifies the approximatechromosomal location of the integrated DNA molecule. For exemplarymethodologies in this regard, see Methods in Plant Molecular Biology andBiotechnology, Glick and Thompson Eds., 269:284, CRC Press, Boca Raton(1993). Map information concerning chromosomal location is useful forproprietary protection of a subject transgenic plant. If unauthorizedpropagation is undertaken and crosses made with other germplasm, the mapof the integration region can be compared to similar maps for suspectplants, to determine if the latter have a common parentage with thesubject plant. Map comparisons would involve hybridizations, RFLP, PCR,SSR, and sequencing, all of which are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

A. Genes that Confer Resistance to Pests or Disease and that Encode:

1. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with a clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., Science, 266:789(1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporiumflavum); Martin, et al., Science, 262:1432 (1993) (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);and Mindrinos, et al., Cell, 78:1089 (1994) (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae).

2. A Bacillus thuringiensis protein, a derivative thereof, or asynthetic polypeptide modeled thereon. See, for example, Geiser, et al.,Gene, 48:109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995, and31998.

3. A lectin. See, for example, the disclosure by Van Damme, et al.,Plant Mol. Biol., 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

4. A vitamin-binding protein such as avidin. See PCT Application No. US93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

5. An enzyme inhibitor, for example, a protease or proteinase inhibitor,or an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem.,262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub, et al., Plant Mol. Biol., 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); and Sumitani,et al., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequenceof Streptomyces nitrosporeus α-amylase inhibitor).

6. An insect-specific hormone or pheromone, such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., Nature, 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

7. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem., 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor) and Pratt, etal., Biochem. Biophys. Res. Comm., 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also, U.S. Pat. No. 5,266,317 toTomalski, et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

8. An insect-specific venom produced in nature, by a snake, a wasp, etc.For example, see Pang, et al., Gene, 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

9. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative,or another non-protein molecule with insecticidal activity.

10. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See PCTApplication No. WO 93/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC under Accession Nos. 39637 and 67152. See also, Kramer, et al.,Insect Biochem. Mol. Biol., 23:691 (1993), who teach the nucleotidesequence of a cDNA encoding tobacco hornworm chitinase, and Kawalleck,et al., Plant Mol. Biol., 21:673 (1993), who provide the nucleotidesequence of the lettuce ubi4-2 polyubiquitin gene.

11. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., Plant Mol. Biol., 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess,et al., Plant Physiol., 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

12. A hydrophobic moment peptide. See PCT Application No. WO 95/16776(disclosure of peptide derivatives of tachyplesin which inhibit fungalplant pathogens) and PCT Application No. WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

13. A membrane permease, a channel former, or a channel blocker. Forexample, see the disclosure of Jaynes, et al., Plant Sci., 89:43 (1993),of heterologous expression of a cecropin-β, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

14. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy, et al., Ann. Rev. Phytopathol.,28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus, and tobacco mosaic virus. Id.

15. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. SeeTaylor, et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions, Edinburgh, Scotland (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

16. A virus-specific antibody. See, for example, Tavladoraki, et al.,Nature, 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

17. A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo-α-1, 4-D-polygalacturonases facilitatefungal colonization and plant nutrient released by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb, et al., Bio/technology,10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubart,et al., Plant J., 2:367 (1992).

18. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., Bio/technology, 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

Any of the above listed disease or pest resistance genes (1-19) can beintroduced into the claimed wild rocket variety through a variety ofmeans including but not limited to transformation and crossing.

B. Genes that Confer Resistance to an Herbicide:

1. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee, etal., EMBO J., 7:1241 (1988) and Miki, et al., Theor. Appl. Genet.,80:449 (1990), respectively.

2. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds, such as glufosinate(phosphinothricin acetyl transferase (PAT), dicamba and Streptomyceshygroscopicus phosphinothricin-acetyl transferase PAT bar genes), andpyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC Accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. See also, Umaballava-Mobapathie in TransgenicResearch, 8:1, 33-44 (1999) that discloses Lactuca sativa resistant toglufosinate. European Patent Application No. 0 333 033 to Kumada, etal., and U.S. Pat. No. 4,975,374 to Goodman, et al., disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides, such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in EuropeanApplication No. 0 242 246 to Leemans, et al. DeGreef, et al.,Bio/technology, 7:61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2, and Acc1-S3 genes described byMarshall, et al., Theor. Appl. Genet., 83:435 (1992).

3. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla, et al.,Plant Cell, 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., Biochem. J.,285:173 (1992).

4. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori, et al., Mol. Gen.Genet., 246:419 (1995). Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., PlantPhysiol., 106:17 (1994)), genes for glutathione reductase and superoxidedismutase (Aono, et al., Plant Cell Physiol., 36:1687 (1995)), and genesfor various phosphotransferases (Datta, et al., Plant Mol. Biol., 20:619(1992)).

5. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306, 6,282,837,5,767,373, and International Publication WO 01/12825.

Any of the above listed herbicide genes (1-5) can be introduced into theclaimed wild rocket variety through a variety of means including, butnot limited to, transformation and crossing.

C. Genes that Confer or Contribute to a Value-Added Trait, Such as:

1. Increased iron content of the wild rocket, for example, byintroducing into a plant a soybean ferritin gene as described in Goto,et al., Acta Horticulturae., 521, 101-109 (2000).

2. Decreased nitrate content of leaves, for example, by introducing intoa wild rocket a gene coding for a nitrate reductase. See, for example,Curtis, et al., Plant Cell Rep., 18:11, 889-896 (1999).

3. Increased sweetness of the wild rocket by introducing a gene codingfor monellin that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See Penarrubia, et al., Bio/technology, 10:561-564 (1992).

4. Modified fatty acid metabolism, for example, by introducing into aplant an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon, et al., PNAS, 89:2625 (1992).

5. Modified carbohydrate composition effected, for example, byintroducing into plants a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza, et al., J. Bacteriol., 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene); Steinmetz, et al., Mol. Gen. Genet., 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene);Pen, et al., Bio/technology, 10:292 (1992) (production of transgenicplants that express Bacillus lichenifonnis α-amylase); Elliot, et al.,Plant Mol. Biol., 21:515 (1993) (nucleotide sequences of tomatoinvertase genes); Søgaard, et al., J. Biol. Chem., 268:22480 (1993)(site-directed mutagenesis of barley α-amylase gene); and Fisher, etal., Plant Physiol., 102:1045 (1993) (maize endosperm starch branchingenzyme II).

D. Genes that Control Male-Sterility:

1. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac-PPT. See International Publication WO 01/29237.

2. Introduction of various stamen-specific promoters. See InternationalPublications WO 92/13956 and WO 92/13957.

3. Introduction of the barnase and the barstar genes. See Paul, et al.,Plant Mol. Biol., 19:611-622 (1992).

Methods for Wild Rocket Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-Mediated Transformation:

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch, et al., Science, 227:1229 (1985); Curtis, et al., Journal ofExperimental Botany, 45:279, 1441-1449 (1994); Torres, et al., PlantCell Tissue and Organ Culture, 34:3, 279-285 (1993); and Dinant, et al.,Molecular Breeding, 3:1, 75-86 (1997). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci., 10:1(1991). Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber, et al.,supra, Miki, et al., supra, and Moloney, et al., Plant Cell Rep., 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer:

Several methods of plant transformation collectively referred to asdirect gene transfer have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereDNA is carried on the surface of microprojectiles measuring 1 μm to 4μm. The expression vector is introduced into plant tissues with abiolistic device that accelerates the microprojectiles to speeds of 300m/s to 600 m/s which is sufficient to penetrate plant cell walls andmembranes. Russell, D. R., et al., Plant Cell Rep., 12 (3, January),165-169 (1993); Aragao, F. J. L., et al., Plant Mol. Biol., 20 (2,October), 357-359 (1992); Aragao, F. J. L., et al., Plant Cell Rep., 12(9, July), 483-490 (1993); Aragao, Theor. Appl. Genet., 93:142-150(1996); Kim, J., Minamikawa, T., Plant Sci., 117:131-138 (1996);Sanford, et al., Part. Sci. Technol., 5:27 (1987); Sanford, J. C.,Trends Biotech., 6:299 (1988); Klein, et al., Bio/technology, 6:559-563(1988); Sanford, J. C., Physiol. Plant, 7:206 (1990); Klein, et al.,Bio/technology, 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/technology, 9:996 (1991).Alternatively, liposome and spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes, et al., EMBO J.,4:2731 (1985) and Christou, et al., PNAS, 84:3962 (1987). Direct uptakeof DNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol, orpoly-L-ornithine has also been reported. Hain, et al., Mol. Gen. Genet.,199:161 (1985) and Draper, et al., Plant Cell Physiol., 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M., Kuhne, T., Biologia Plantarum, 40(4):507-514(1997/98); Donn, et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin, etal., Plant Cell, 4:1495-1505 (1992); and Spencer, et al., Plant Mol.Biol., 24:51-61 (1994). See also Chupean, et al., Bio/technology, 7:5,503-508 (1989).

Following transformation of wild rocket target tissues, expression ofthe above-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossedwith another (non-transformed or transformed) line in order to produce anew transgenic wild rocket line. Alternatively, a genetic trait whichhas been engineered into a particular wild rocket variety using theforegoing transformation techniques could be introduced into anotherline using traditional backcrossing techniques that are well known inthe plant breeding arts. For example, a backcrossing approach could beused to move an engineered trait from a public, non-elite inbred lineinto an elite inbred line, or from an inbred line containing a foreigngene in its genome into an inbred line or lines which do not containthat gene. As used herein, “crossing” can refer to a simple X by Ycross, or the process of backcrossing, depending on the context.

Gene Conversions

When the term “wild rocket plant” is used in the context of the presentinvention, this also includes any gene conversions of that variety. Theterm “gene converted plant” as used herein refers to those wild rocketplants which are developed by backcrossing, genetic engineering, ormutation, where essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition tothe one or more genes transferred into the variety via the backcrossingtechnique, genetic engineering, or mutation. Backcrossing methods can beused with the present invention to improve or introduce a characteristicinto the variety. The term “backcrossing” as used herein refers to therepeated crossing of a hybrid progeny back to the recurrent parent,i.e., backcrossing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times to therecurrent parent. The parental wild rocket plant which contributes thegene for the desired characteristic is termed the “nonrecurrent” or“donor parent.” This terminology refers to the fact that thenonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental wild rocket plant to which thegene or genes from the nonrecurrent parent are transferred is known asthe recurrent parent as it is used for several rounds in thebackcrossing protocol. Poehlman & Sleper (1994) and Fehr (1993). In atypical backcross protocol, the original variety of interest (recurrentparent) is crossed to a second variety (nonrecurrent parent) thatcarries the gene of interest to be transferred. The resulting progenyfrom this cross are then crossed again to the recurrent parent and theprocess is repeated until a wild rocket plant is obtained whereessentially all of the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, in addition to the transferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a trait or characteristic in the original line.To accomplish this, a gene of the recurrent variety is modified orsubstituted with the desired gene from the nonrecurrent parent, whileretaining essentially all of the rest of the desired genetic, andtherefore the desired physiological and morphological, constitution ofthe original line. The choice of the particular nonrecurrent parent willdepend on the purpose of the backcross. One of the major purposes is toadd some commercially desirable, agronomically important trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many gene traits have been identified that are not regularly selected inthe development of a new line but that can be improved by backcrossingtechniques. Gene traits may or may not be transgenic. Examples of thesetraits include, but are not limited to, male sterility, modified fattyacid metabolism, modified carbohydrate metabolism, herbicide resistance,resistance for bacterial, fungal, or viral disease, insect resistance,enhanced nutritional quality, industrial usage, yield stability, andyield enhancement. These genes are generally inherited through thenucleus. Several of these gene traits are described in U.S. Pat. Nos.5,777,196, 5,948,957, and 5,969,212, the disclosures of which arespecifically hereby incorporated by reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of wild rocket andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Teng, et al., HortScience, 27:9,1030-1032 (1992); Teng, et al., HortScience, 28:6, 669-1671 (1993);Zhang, et al., Journal of Genetics and Breeding, 46:3, 287-290 (1992);Webb, et al., Plant Cell Tissue and Organ Culture, 38:1, 77-79 (1994);Curtis, et al., Journal of Experimental Botany, 45:279, 1441-1449(1994); Nagata, et al., Journal for the American Society forHorticultural Science, 125:6, 669-672 (2000); and Ibrahim, et al., PlantCell Tissue and Organ Culture, 28(2), 139-145 (1992). It is clear fromthe literature that the state of the art is such that these methods ofobtaining plants are routinely used and have a very high rate ofsuccess. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce wild rocket plants havingthe physiological and morphological characteristics of variety‘Prudenzia’ and ‘Anastazia’.

As used herein, the term “tissue culture” indicates a compositioncontaining isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, suckers, and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture containing organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

Additional Breeding Methods

The invention is also directed to methods for producing a wild rocketplant by crossing a first parent wild rocket plant with a second parentwild rocket plant where the first or second parent wild rocket plant isa wild rocket plant of variety ‘Prudenzia’ or ‘Anastazia’. Further, bothfirst and second parent wild rocket plants can come from wild rocketvariety ‘Prudenzia’ or ‘Anastazia’. Thus, any such methods using wildrocket variety ‘Prudenzia’ or ‘Anastazia’ are part of the invention:selfing, backcrosses, hybrid production, crosses to populations, and thelike. All plants produced using wild rocket variety ‘Prudenzia’ or‘Anastazia’ as at least one parent are within the scope of thisinvention, including those developed from varieties derived from wildrocket variety ‘Prudenzia’ or ‘Anastazia’. Advantageously, this wildrocket variety could be used in crosses with other, different, wildrocket plants to produce the first generation (F₁) wild rocket hybridseeds and plants with superior characteristics. The variety of theinvention can also be used for transformation where exogenous genes areintroduced and expressed by the variety of the invention. Geneticvariants created either through traditional breeding methods using wildrocket variety ‘Prudenzia’ or ‘Anastazia’ or through transformation ofvariety ‘Prudenzia’ or ‘Anastazia’ by any of a number of protocols knownto those of skill in the art are intended to be within the scope of thisinvention.

The following describes breeding methods that may be used with wildrocket variety ‘Prudenzia’ or ‘Anastazia’ in the development of furtherwild rocket plants. One such embodiment is a method for developingvariety ‘Prudenzia’ or ‘Anastazia’ progeny wild rocket plants in a wildrocket plant breeding program, by: obtaining the wild rocket plant, or apart thereof, of variety ‘Prudenzia’ or ‘Anastazia’, utilizing saidplant or plant part as a source of breeding material, and selecting awild rocket variety ‘Prudenzia’ or ‘Anastazia’ progeny plant withmolecular markers in common with variety ‘Prudenzia’ or ‘Anastazia’and/or with morphological and/or physiological characteristics selectedfrom the characteristics listed in the section entitled “Objectivedescription of the variety ‘Prudenzia’” or “Objective description of thevariety ‘Anastazia’”. Breeding steps that may be used in the wild rocketplant breeding program include pedigree breeding, backcrossing, mutationbreeding, and recurrent selection. In conjunction with these steps,techniques such as RFLP-enhanced selection, genetic marker enhancedselection (for example, SSR markers), and the making of double haploidsmay be utilized.

Another method involves producing a population of wild rocket variety‘Prudenzia’ or ‘Anastazia’ progeny wild rocket plants, by crossingvariety ‘Prudenzia’ or ‘Anastazia’ with another wild rocket plant,thereby producing a population of wild rocket plants, which, on average,derive 50% of their alleles from wild rocket variety ‘Prudenzia’ or‘Anastazia’. A plant of this population may be selected and repeatedlyselfed or sibbed with a wild rocket variety resulting from thesesuccessive filial generations. One embodiment of this invention is thewild rocket variety produced by this method and that has obtained atleast 50% of its alleles from wild rocket variety ‘Prudenzia’ or‘Anastazia’. One of ordinary skill in the art of plant breeding wouldknow how to evaluate the traits of two plant varieties to determine ifthere is no significant difference between the two traits expressed bythose varieties. For example, see Fehr and Walt, Principles of VarietyDevelopment, pp. 261-286 (1987). Thus the invention includes wild rocketvariety ‘Prudenzia’ or ‘Anastazia’ progeny wild rocket plants containinga combination of at least two variety ‘Prudenzia’ or ‘Anastazia’ traitsselected from those listed in the section entitled “Objectivedescription of the variety ‘Prudenzia’” or “Objective description of thevariety ‘Anastazia’”; or the variety ‘Prudenzia’ or ‘Anastazia’combination of traits listed in the Summary of the Invention, so thatsaid progeny wild rocket plant is not significantly different for saidtraits than wild rocket variety ‘Prudenzia’ or ‘Anastazia’ as determinedat the 5% significance level when grown in the same environmentalconditions. Using techniques described herein, molecular markers may beused to identify said progeny plant as a wild rocket variety ‘Prudenzia’or ‘Anastazia’ progeny plant. Mean trait values may be used to determinewhether trait differences are significant, and preferably the traits aremeasured on plants grown under the same environmental conditions. Oncesuch a variety is developed, its value is substantial since it isimportant to advance the germplasm base as a whole in order to maintainor improve traits such as yield, disease resistance, pest resistance,and plant performance in extreme environmental conditions.

Progeny of wild rocket variety ‘Prudenzia’ or ‘Anastazia’ may also becharacterized through their filial relationship with wild rocket variety‘Prudenzia’ or ‘Anastazia’, as for example, being within a certainnumber of breeding crosses of wild rocket variety ‘Prudenzia’ or‘Anastazia’. A breeding cross is a cross made to introduce new geneticsinto the progeny, and is distinguished from a cross, such as a self or asib cross, made to select among existing genetic alleles. The lower thenumber of breeding crosses in the pedigree, the closer the relationshipbetween wild rocket variety ‘Prudenzia’ or ‘Anastazia’ and its progeny.For example, progeny produced by the methods described herein may bewithin 1, 2, 3, 4, or 5 breeding crosses of wild rocket variety‘Prudenzia’ or ‘Anastazia’.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which wild rocket plantscan be regenerated, plant calli, plant clumps, and plant cells that areintact in plants or parts of plants, such as leaves, pollen, embryos,cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers,ovules, seeds, stems, and the like.

The use of the terms “a,” “an,” and “the,” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions, and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

DEPOSIT INFORMATION Wild Rocket Variety ‘Prudenzia’

A deposit of the wild rocket variety ‘Prudenzia’ is maintained by EnzaZaden USA, Inc., having an address at 7 Harris Place, Salinas, Calif.93901, United States. Access to this deposit will be available duringthe pendency of this application to persons determined by theCommissioner of Patents and Trademarks to be entitled thereto under 37C.F.R. §1.14 and 35 U.S.C. §122. Upon allowance of any claims in thisapplication, all restrictions on the availability to the public of thevariety will be irrevocably removed by affording access to a deposit ofat least 2,500 seeds of the same variety with the National Collection ofIndustrial, Food and Marine Bacteria Ltd. (NCIMB Ltd), FergusonBuilding, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, UnitedKingdom.

At least 2500 seeds of wild rocket variety ‘Prudenzia’ were deposited onDATE according to the Budapest Treaty in the National Collection ofIndustrial, Food and Marine Bacteria Ltd (NCIMB Ltd), Ferguson Building,Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, United Kingdom. Thedeposit has been assigned NCIMB number X1. Access to this deposit willbe available during the pendency of this application to personsdetermined by the Commissioner of Patents and Trademarks to be entitledthereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. Upon allowance of anyclaims in this application, all restrictions on the availability to thepublic of the variety will be irrevocably removed.

The deposit will be maintained in the NCIMB depository, which is apublic depository, for a period of at least 30 years, or at least 5years after the most recent request for a sample of the deposit, or forthe effective life of the patent, whichever is longer, and will bereplaced if a deposit becomes nonviable during that period.

Wild Rocket Variety ‘Anastazia’

A deposit of the wild rocket variety ‘Anastazia’ is maintained by EnzaZaden USA, Inc., having an address at 7 Harris Place, Salinas, Calif.93901, United States. Access to this deposit will be available duringthe pendency of this application to persons determined by theCommissioner of Patents and Trademarks to be entitled thereto under 37C.F.R. §1.14 and 35 U.S.C. §122. Upon allowance of any claims in thisapplication, all restrictions on the availability to the public of thevariety will be irrevocably removed by affording access to a deposit ofat least 2,500 seeds of the same variety with the National Collection ofIndustrial, Food and Marine Bacteria Ltd. (NCIMB Ltd), FergusonBuilding, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, UnitedKingdom.

At least 2500 seeds of wild rocket variety ‘Anastazia’ were deposited onDATE according to the Budapest Treaty in the National Collection ofIndustrial, Food and Marine Bacteria Ltd (NCIMB Ltd), Ferguson Building,Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, United Kingdom. Thedeposit has been assigned NCIMB Number X2. Access to this deposit willbe available during the pendency of this application to personsdetermined by the Commissioner of Patents and Trademarks to be entitledthereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. Upon allowance of anyclaims in this application, all restrictions on the availability to thepublic of the variety will be irrevocably removed.

The deposit will be maintained in the NCIMB depository, which is apublic depository, for a period of at least 30 years, or at least 5years after the most recent request for a sample of the deposit, or forthe effective life of the patent, whichever is longer, and will bereplaced if a deposit becomes nonviable during that period.

1. Wild rocket seed selected from the group consisting of wild rocketseed designated as ‘Prudenzia’, representative sample of seed havingbeen deposited under NCIMB Accession Number X1; and wild rocket seeddesignated as ‘Anastazia’, representative sample of seed having beendeposited under NCIMB Accession Number X2.
 2. A wild rocket plantproduced by growing the seed of claim
 1. 3. A plant part from the plantof claim
 2. 4. The plant part of claim 3 wherein said part is a stem, aleaf, or a portion thereof.
 5. A wild rocket plant having all thephysiological and morphological characteristics of the wild rocket plantof claim
 2. 6. A plant part from the plant of claim
 5. 7. The plant partof claim 6, wherein said part is a stem, a leaf, or a portion thereof.8. An F₁ hybrid wild rocket plant having ‘Prudenzia’ as a parent where‘Prudenzia’ is grown from the seed of claim
 1. 9. An F₁ hybrid wildrocket plant having ‘Anastazia’ as a parent where ‘Anastazia’ is grownfrom the seed of claim
 1. 10. A pollen grain or an ovule of the plant ofclaim
 2. 11. A tissue culture of the plant of claim
 2. 12. A wild rocketplant regenerated from the tissue culture of claim 11, wherein the planthas all of the morphological and physiological characteristics of a wildrocket plant produced by growing seed designated as ‘Prudenzia’,representative sample of seed having been deposited under having NCIMBAccession Number X1.
 13. A wild rocket plant regenerated from the tissueculture of claim 11, wherein the plant has all of the morphological andphysiological characteristics of a wild rocket plant produced by growingseed designated as ‘Anastazia’, representative sample of seed havingbeen deposited under having NCIMB Accession Number X2.
 14. A method ofmaking wild rocket seeds, said method comprising crossing the plant ofclaim 2 with another wild rocket plant and harvesting seed therefrom.15. A method of making wild rocket variety ‘Prudenzia’, said methodcomprising selecting seeds from the cross of one ‘Prudenzia’ plant withanother ‘Prudenzia’ plant, a sample of ‘Prudenzia’ wild rocket seedhaving been deposited under NCIMB Accession Number X1.
 16. A method ofmaking wild rocket variety ‘Anastazia’, said method comprising selectingseeds from the cross of one ‘Anastazia’ plant with another ‘Anastazia’plant, a sample of ‘Anastazia’ wild rocket seed having been depositedunder NCIMB Accession Number X2.